As we are heading towards the high season of flu I though it would be interesting to remember how the vaccines against influenza virus (the causative agent of flu) are actually made.

Worldwide there are 2 flu seasons one in the Northern hemisphere (October- May) and another in the Southern hemisphere (May- October). Egg-based vaccine production is the most common way that the vaccines against the seasonal flu are made. It takes around 6 months from the initial prediction of the next season’s flu strain to a flu shot in the doctor’s office and around 900 million eggs are required to make 300 million doses of flu vaccine each year (I initially though that it was a lot of eggs but then considering that 1.8 trillion eggs are consumed globally each year I suppose that wouldn’t even show on the graph of global egg consumption per month ).

The prediction of the next season’s flu strains is based on the surveillance data of the strains circulating in the population, if a different type of strain appears then the components of the flu shot are modified accordingly. For the 2014-2015 flu season the vaccines include antigens for different strains of H1N1, H3N2 and Influenza B viruses.

The preparation of vaccines in eggs might sound a bit wonky but it goes quite a while back. A number of viruses can be grown in eggs and the first egg culture based vaccine was made in 1935 to vaccinate people against smallpox. After the first identification of influenza virus as the causative agent of seasonal flu (in 1933 by W. Smith, C.H. Andrewes and P. Laidlaw) in 1938 Thomas Francis and Jonas Stalk (of the polio vaccine fame) were the first to produce a vaccine against flu and though, initially it was only given to US military fighting in the WWII, it also became available to the public in 1950s.

The process of vaccine production in eggs is quite fascinating. Influenza virus has a genome that is divided into 8 segments and each segment codes for one or more proteins. Two proteins, hemagglutinin (HA) and neuraminidase (NA) are used for vaccine production because they are good antigens for eliciting immune response. For production of large amounts of HA and NA from different viral strains the sequences of these proteins for each strain need to be determined. Then the sequences are inserted into a lab adapted influenza strain called PR8 so what you get is a PR8 virus in which native HA and NA genes are changed to HA and NA that belong to the seasonal flu strains. The PR8 strain is used mainly because it grows well in eggs and therefore large quantities of the proteins can be made and also for safety reasons because it would be quite dangerous to produce large quantities of seasonal or pandemic strains against which many people have little or no immunity.

The PR8 hybrid is then injected into 12 day-old embryonated chicken eggs where it grows and multiplies for 3 days and after 3 days the allantoic fluid of the egg essentially becomes a viral soup. The allantoic fluid is harvested, the virus in it is exposed to chemicals that kill it, and HA and NA proteins are then purified to be used for vaccination.

A technique called candling is used to see if any of the virus-containing eggs are cracked or contaminated in which case they are removed from the process

Obviously egg-based production is quite limited in terms of the quantities of the antigen obtained and the time the whole production process takes. It’s not a problem for the seasonal strains but the pandemic strains that usually appear unexpectedly reveal these limitations quite clearly (as was seen for 2009 H1N1 pandemic during which vaccine shortage cause considerable concerns in many countries). Consequently, a substantial effort has been placed on development of new ways to produce vaccines. In 2012, for example, FDA and its equivalents approved a vaccine called FlucelVax, which was the first influenza vaccine to be made in animal cell cultures.

However, the innovation didn’t stop there and in response to H1N1 pandemic the U.S. Defence Advanced Research Projects Agency (DARPA) has began to develop a plant-based vaccine production system. Personally, the concept of vaccines made in plants sounds even stranger than egg-based production for me, but that’s, I suppose, is my first-impression speaking, on the other hand the scientists in me says ‘Awesome! Let’s so it!’.

The tobacco plant was chosen for vaccine production because it is one of the model lab organisms (meaning it’s a species that we know a lot about at macro and micro scales, which makes it easier to work with in the lab). At the same time at least it’s one good commercial use for these plants and maybe some tobacco companies will have something to do once people understand how stupid smoking is (not that I necessary think that will ever happen).

Cultivating tobacco plants for vaccines.

The vaccine made in plants is still based on HA protein, however, rather than expressing it in eggs it is made by the plant cells. The plant can either be permanently transformed to include an HA gene in its DNA so that it and subsequent generations would be producing the protein, or it can be infected and populated with a bacteria that carry a Cauliflower Mosaic Virus (CaMV). CaMV has HA gene incorporated in it and as the virus spreads and multiplies in the plant the HA protein is made as well. The process of infection and production of HA only takes a couple of weeks so it would speed up vaccine production greatly in case of a pandemic outbreak. Various experiments have shown that it is possible to produce anywhere between 200 and 1300 milligrams of HA protein per one kilogram of tobacco leaves and considering that one vaccine dose requires 15 micrograms of the antigen per strain this is a highly efficient process. Moreover, several plant-based vaccines have now entered phase one and two human trials and have showed promising results.

Interestingly, the infamous Zmapp, a monoclonal antibody based drug against Ebola is also made in tobacco plants by Kentucky BioProcessing firm which belongs to the second largest tobacco company in U.S. Reynolds American, Inc.